Charles F. Aylsworth

Counteraction of gonadal steroid inhibition of luteinizing hormone release by naloxone.

The effect of naloxone on the negative feedback action of gonadal steroids on luteinizing hormone (LH) release was studied in castrated male and female rats. The reduction by estradiol benzoate or testosterone propionate of elevated serum LH levels in rats ovariectomized for 4 weeks was reversed by a single injection of naloxone. Injection of estradiol benzoate, together with progesterone, similarly reduced serum LH levels in ovariectomized rats, and this inhibition was partially reversed by naloxone. A single injection of testosterone propionate decreased serum LH levels in male rats castrated 48 h earlier, and naloxone completely blocked the androgen-induced inhibition of LH secretion. Chronic administration of either testosterone propionate or morphine sulfate to castrated male rats prevented the postcastration rise of serum LH. The decrease of hypothalamic LHRH in castrated rats was completely blocked by testosterone propionate or morphine sulfate administration. These results are believed to indicate that hypothalamic opiates are involved in gonadal steroid feedback inhibition of LH release.

Evidence for Hypothalamic Noradrenergic Involvement in Naloxone-Induced Stimulation of Luteinizing Hormone Release

A single injection of the opiate antagonist, naloxone (NAL), resulted in a fourfold increase in serum luteinizing hormone (LH) concentration 20 min after injection. To determine whether noradrenergic neurons were involved, male Sprague-Dawley rats were treated with alpha-methyl-p-tyrosine (alpha-MPT), phenoxybenzamine hydrochloride (PBH), or diethyldithiocarbamate (DDC), all anti-noradrenergic drugs. Reduction of hypothalamic norepinephrine synthesis by alpha-MPT or DDC, or blockade of the alpha-receptors by PBH, resulted in complete suppression of NAL-induced LH release. These results suggest that the NAL-induced increase in LH release is mediated in part via a hypothalamic noradrenergic mechanism.

Effects of a Prolactin-Secreting Pituitary Tumor on Hypothalamic, Gonadotropic and Testicular Function in Male Rats

The presence of a transplanted prolactin- and GH-secreting pituitary tumor (Furth MtT.215) in inbred male rats resulted in increased hypothalamic LHRH and pituitary LH content, decreased serum LH and testosterone concentrations, and very high serum prolactin values. The pituitary tumor also inhibited LH release by the in situ pituitary in response to orchidectomy, or orchidectomy, or adrenalectomy, and reduced the LH response to LHRH administration. Testes weight was significantly reduced and adrenal weight was significantly increased in the rats carrying pituitary tumors. These results that inhibitory effects of the transplanted pituitary tumor on LH and testosterone secretion were affected both a reduction in hypothalamic LHRH release and a reduced responsiveness of the pituitary to LHRH.

Opiate antagonists can inhibit mammary tumor growth in rats.

Summary Female rats with carcinogen-induced mammary adenocarcinomas were injected 2-3 times daily with naloxone or naltrexone, specific antagonists of endogenous opioid peptides. Both drugs completely prevented mammary tumor growth without inducing regression of the cancers, effects believed to be due to reduced prolactin and perhaps GH secretion. These results suggest a possible role for endogenous opioid peptides in rat mammary tumor growth.

Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of ankylosing spondylitis reduce HLA-B27 mediated presentation of multiple antigens

Abstract Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system.

Protein from intestinal Eimeria protozoan stimulates IL‐12 release from dendritic cells, exhibits antitumor properties in vivo and is correlated with low intestinal tumorigenicity

The small intestine (SI) of vertebrates exhibits low tumorigenesis and rarely supports metastatic growth from distant tumors. Many theories have been proposed to address this phenomenon, but none has been consistently supported. One candidate mechanism is that the vast immunologic compartment of the SI provides a heightened level of tumor immunosurveillance. Consistent with this, we have identified a molecule of low abundance from bovine SI that has the hallmarks of a potent immunostimulant and may be associated with the natural suppression of cancer in the intestinal tract. The protein originates from an endemic gut protozoan, Eimeria spp., and is homologous to the antigen 3‐1E previously isolated from the avian apicomplexan E. acervulina. We show here that it is a very potent stimulator of IL‐12 release from dendritic cells, upregulates inflammatory modulators in vivo (IL‐12, MCP‐1, IL‐6, TNF‐α and INF‐γ) and has antitumor properties in mice. In addition, it is synergistic in vitro with anti‐CD40 antibody, IFN‐γ, IL‐4 and GM‐CSF; is active across species barriers in vivo; and has no observable toxicity. Based on these activities, we speculate that it is an inducer of protozoan‐targeted innate immunity, which may explain its potential benefit to the intestinal tract and potency as an agent in cancer immunotherapy.

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: Implication of a role for protein kinase C in the regulation of gap junctional intercellular communication

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6t-hioguanine-resistant (6-TGR) and 6-thioguanine-sensitive (6-TGS) V79 cells significantly increased the recovery of 6-TGR cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TGR cells was significantly greater than the expected (calculated) additive 6-TGR cell recovery. No synergistic increases in 6-TGR cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.

Effects of fatty acids on gap junctional communication: possible role in tumor promotion by dietary fat.

Dietary lipids, in particular unsaturated fat, promote the development of many experimental tumors. However, no mechanisms to fully explain these effects have been elucidated. Recent reports, which we summarize here, suggest a role for gap junction-mediated intercellular communication in the process of tumor promotion. We also review tumor-promoting effects of dietary fat on experimental, particularly mammary, carcinogenesis. Our main focus is to review recent data examining the inhibitory effects of unsaturated fatty acids on metabolic cooperation in Chinese hamster V79 cells. These data suggest that inhibition of junctional communication may be involved mechanistically in the promotion of tumors by high levels of dietary unsaturated fat. Finally, potential mechanisms by which unsaturated fatty acids inhibit metabolic cooperation are examined.

Possible Involvement of Arachidonate Products in Tumor Promoter Inhibition of Cell-Cell Communication

The complex, healthy multicellular organism is the ultimate product of a hierarchy of levels of organization, each interacting with each other and with the environment via different kinds of feedback or communicating mechanisms (1,2). Unless there is a functional hierarchical organization of the molecular, biochemical, cellular, physiological and psychological (in the case of human beings), this state of health cannot be maintained. Proper genetic information in the form of DNA molecules and normal expression of the genetic information are needed to affect the appropriate adaptive biochemistry. Normal regulation of cell proliferation and differentiation is necessary for healthy growth and function of organ systems (3). Proper function of organ systems, in turn, leads to adaptive responses on the whole organism level. Consequently, interference with any of these levels can influence the functions of levels above and below, because of interacting connections between the levels (1,2).

Autoimmune Disease-Associated Variants of Extracellular Endoplasmic Reticulum Aminopeptidase 1 Induce Altered Innate Immune Responses by Human Immune Cells

Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we demonstrated that ERAP1 regulates key aspects of the innate immune response. Previous studies show ERAP1 to be endoplasmic reticulum-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating the innate immune responses of human peripheral blood mononuclear cells (hPBMCs) using two experimental methods: extracellular exposure of hPBMCs to ERAP1 variants and adenovirus (Ad)-based ERAP1 expression. We found that exposure of hPBMCs to ERAP1 variant proteins as well as ERAP1 overexpression by Ad5 vectors increased inflammatory cytokine and chemokine production, and enhanced immune cell activation. Investigating the molecular mechanisms behind these responses revealed that ERAP1 is able to activate innate immunity via multiple pathways, including the NLRP3 (NOD-like receptor, pyrin domain-containing 3) inflammasome. Importantly, these responses varied if autoimmune disease-associated variants of ERAP1 were examined in the assay systems. Unexpectedly, blocking ERAP1 cellular internalization augmented IL-1β production. To our knowledge, this is the first report identifying ERAP1 as being involved in modulating innate responses of human immune cells, a finding that may explain why ERAP1 has been genetically associated with several autoimmune diseases.